Gentamicin uptake by LLCPK1 cells: effect of intracellular and extracellular pH changes

The mechanisms by which aminoglycosides are transported across the luminal membrane of renal proximal tubular cells remain unclear. A luminal organic cation/H+ exchange as well as an adsorptive endocytosis membrane process has been proposed to be involved in gentamicin renal accumulation. The objectives of this work were to explore further the effects of intracellular and extracellular pH changes on gentamicin uptake. [3H]Gentamicin uptake by a continuous renal epithelial cell line, LLCPK1, grown as a monolayer on an impermeable surface was measured at different temperatures and pH conditions and in the presence of various inhibitors. Uptake of gentamicin was found to be carrier mediated (K(m) = 1.26 +/- 0.22 mM, Vmax = 289 +/- 27 pmol.mg-1.min-1), energy dependent (inhibited in part by sodium azide), and temperature dependent (37 degrees C > 4 degrees C). Fifteen-minute gentamicin (10 microM) uptake was inhibited by 1 mM of the organic cations cimetidine (61.0%), quinidine (73.5%), quinine (68.6%), and verapamil (61.5%). More importantly, while an outwardly directed proton gradient did not have a significant effect on gentamicin uptake, extracellular acidification (pH 6.5), which leads to a higher degree of gentamicin ionization, significantly enhanced gentamicin uptake by LLCPK1 monolayer cells. These results suggest that the luminal organic cation/H+ exchanger is not involved in gentamicin uptake by renal cultured epithelial cells. Rather, the cationic charge of gentamicin appears to be one of the primary determinants for renal luminal uptake.